The goal of this proposal is to determine the structural basis by which G protein-coupled receptors (GPCRs) activate specific G proteins. The majority of hormones and neurotransmitters communicate information to cells via GPCRs, and GPCRs represent the largest group of targets for drug development. Our laboratories have a long-standing interest in elucidating the structure and mechanism of G protein activation by GPCRs. During the previous funding period we succeeded in obtaining the first crystal structure of a GPCR-G protein complex: the beta2 adrenoceptor (?2AR) in complex with Gs, the stimulatory protein for adenylyl cyclase. This structure provides important mechanistic insight into G protein activation, but at the same time raises new questions that will be addressed in this competitive renewal. Specific Aims include: Aim 1. Determine the structural basis of GPCR-G protein coupling specificity. The structure of the ?2AR-Gs complex provided the first high-resolution insights into transmembrane signaling by a GPCR. However, additional GPCR-G protein complexes will be required to understand the structural basis for G protein coupling specificity, and to determine if the mechanistic insights obtained from the ?2AR -Gs structure are generalizable to other GPCR-G protein pairs. We therefore propose to obtain three additional GPCR-G protein complex structures: (1) the vasopressin receptor-Gs complex; (2) the structure of the ?2AR-Gi complex; and (3) the structure of the M2R-Gi complex. Aim 2. Characterize the formation of the ?2AR-Gs complex from the GDP bound Gs heterotrimer. The ?2AR- Gs crystal structure represents a single state in a complex cycle of events. The process of complex formation and dissociation remains poorly understood. These are dynamic process that may not be addressable by crystallography; however, the ?2AR-Gs structure will provide the basis for designing and interpreting biochemical and biophysical studies to characterize the mechanism of complex formation and dissociation. In Aim 2 we will characterize the low affinity interactions between the ?2AR and GDP bound Gs. These interactions may play a role in G protein coupling specificity. Aim 3. Characterize the process of ?2AR -Gs dissociation following GTP binding. The goal of this Aim is to understand how the ?2AR -Gs complex dissociates into active signaling proteins upon binding GTP and to identify persisting interactions between any of the three components: ?2AR, G?s and G??. Aim 4. Characterize the dynamic behavior of the G?s alpha helical domain. The most surprising and unexpected feature of ?2AR-Gs structures is the flexible link between the two domains that make up G?s: the Ras-like GTPase domain and the alpha helical domain (AHD). This subaim will further characterize the interactions between these two domains in the ?2AR-Gs complex as well as in GTP and GDP bound states.